Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses

Abstract

The purpose of this study was to compare the biochemical and biological properties of nonstructural protein (nsp) 15 among mouse hepatitis virus (MHV), severe acute respiratory syndrome coronavirus (SARS-CoV) and transmissible gastroenteritis virus (TGEV) in virus-infected and ectopically expressed cells. In virus-infected cells, MHV nsp15 distributed unevenly throughout the cytoplasm but predominantly in the perinuclear region. When expressed as N-terminal enhanced green fluorescence protein (EGFP) fusion, it predominantly formed speckles in the cytoplasm. In contrast, SARS-CoV and TGEV EGFP-nsp15s distributed smoothly in the whole cell and did not form speckles. Deletion mapping experiments identified two domains responsible for the speckle formation in MHV EGFP-nsp15: Domain I (aa101–150) and Domain III (aa301–374). Interestingly, Domain II (aa151–250) had an inhibitory effect on Domain III- but not Domain I-mediated speckle formation. Expression of a small (35aa) sequence in Domain III alone was sufficient to form speckles for all 3 viral nsp15s. However, addition of surrounding sequences in Domain III abolished the speckle formation for TGEV nsp15 but not for MHV and SARS-CoV nsp15s. Further domain swapping experiments uncovered additional speckle-inducing and -suppressive elements in nsp15s of SARS-CoV and TGEV. Homotypic interaction involving Domain III of MHV nsp15 was further demonstrated biochemically. Moreover, the biological functions of the expressed nsp15s were assessed in MHV-infected cells. It was found that the effects of EGFP-nsp15s on MHV replication were both virus species- and nsp15 domain-dependent. Collectively these results thus underscore the differential biochemical and biological functions among the nsp15s of MHV, TGEV and SARS-CoV in host cells.

A screen of the NIH Clinical Collection small molecule library identifies potential anti-coronavirus drugs

“…For detecting proteins, either Western blot analysis or IFA was performed as previously described (Cao and Zhang, 2012).…”

To sense or not to sense viral RNA—essentials of coronavirus innate immune evasion

“…However, the discovery of an insect-born Nidovirus and the re-evaluation of invertebrate Ronivirus genomes revealed that they lack an EndoU domain, indicating that the utilization of an EndoU domain is restricted to vertebrate Nidoviruses [6 ]. The CoV-EndoU domain is structurally very conserved but exhibits only moderate sequence conservation [40,41]. It hydrolyses ssRNA and dsRNA substrates, preferably 3 0 of uridylates [41,42] and cleaves similar as XendoU and RNase A, as the reaction products possess 2 0 ,3 0 -cyclic phosphate ends [42].…”

“…It hydrolyses ssRNA and dsRNA substrates, preferably 3 0 of uridylates [41,42] and cleaves similar as XendoU and RNase A, as the reaction products possess 2 0 ,3 0 -cyclic phosphate ends [42]. Ectopic expression studies demonstrated that CoV-nsp15 is not only colocalized with the RTC, but also distributed throughout the cytoplasm [40], thus an additional function besides its involvement in viral replication has been proposed [43].…”

Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Suppresses Both MAVS and RIG-I Expression as One of the Mechanisms to Antagonize Type I Interferon Production

“…PRRSV nsp11 has the uridylatepreference cleavage of RNA and consists of two subdomains, subdomain A and subdomain B. Subdomain A is thought to maintain the nuclease activity while subdomain B may be important for overall structural conformation. From its newly determined crystal structure, nsp11 assembles into an asymmetric dimmer [22], which differs from the hexametric structure of coronaviruses nsp15 [29,30]. However, enzymatic sites of PRRSV nsp11 are perfectly superimposed on coronavirus nsp15 with two His residues in the "active loop" and one Val residue and one Thr residue in the "supportive loop" [31][32][33][34].…”